The Anatomical Origins of Migratory Dendritic Cells in the Intestine Stephanie Ailsa Houston BSc ...

273 Pages·2014·11.36 MB·English

by  Stephanie Houston

Houston, Stephanie Ailsa (2013) The anatomical origins of migratory
dendritic cells in the intestine. PhD thesis.

http://theses.gla.ac.uk/5265/

Copyright and moral rights for this work are retained by the author
A copy can be downloaded for personal non-commercial research or study, without prior
permission or charge
This work cannot be reproduced or quoted extensively from without first obtaining
permission in writing from the author
The content must not be changed in any way or sold commercially in any format or
medium without the formal permission of the author
When referring to this work, full bibliographic details including the author, title,
awarding institution and date of the thesis must be given

Enlighten:Theses
http://theses.gla.ac.uk/
[email protected]
The Anatomical Origins of Migratory Dendritic
Cells in the Intestine

Stephanie Ailsa Houston
BSc, MRes

Submitted in fulfilment of the requirement for the degree of
Doctor of Philosophy

Institute of Infection, Immunity and Inflammation
College of Medical Veterinary and Life Sciences
University of Glasgow

December 2013
Abstract
The intestine is exposed to a vast array of harmless dietary antigen as well as an
enormous community of commensal bacteria. Alongside this harmless antigen,
pathogens can enter the body via the intestinal mucosal surface. The intestinal
immune system must discriminate between pathogens and innocuous antigens.
Dendritic cells (DCs), present in the small intestine (SI) and colon, are
fundamental in controlling intestinal immune responses; they migrate to the
mesenteric lymph node (MLN) and prime effector or regulatory T cells.
Furthermore, DCs direct the immune response in the gut-associated lymphoid
tissues (GALT), the Peyer’s patches (PP) and isolated lymphoid follicles (ILFs).
The aim of this work was to determine the anatomical origins of DCs in the MLN.
First, the anatomical organisation of lymphatic vessels draining to the MLN from
the SI and colon was investigated. Second, DC migration in mice lacking specific
GALT was explored. Finally, the migration of DCs from PPs to the MLN was
investigated. To achieve these aims, DC migration was studied using a variety of
labelling methods, mice that lacked specific GALT were employed, and surgical
procedures were used to collect DCs from the thoracic duct.
Here, I demonstrate the anatomical segregation of DCs that migrate to the MLN
from the SI and colon. This was reflected in differences in DC subset composition
and antigen presentation in the SI and colon-draining nodes of the MLN. Separate
analysis of MLN nodes will allow a more refined understanding of intestinal
immune responses. All but one DC subset, CD103-CD11b- DCs, were still present
in pseudo-afferent lymph from mice lacking both PPs and ILFs. This subset is
therefore likely to originate from either PPs or ILFs. Surprisingly, CD103+CD8α+
DCs were present in these mice, showing that many CD8α+ DCs were resident in
the lamina propria and are not limited to lymphoid tissue. Four DC subsets are
able to migrate from the intestine in PP-null mice, suggesting that CD103-CD11b-
DCs migrate specifically from ILFs. I then demonstrated that DCs migrate from
PPs to the MLN. These migrating PP DCs expanded in response to a DC specific
growth factor and their migration depended on CCR7 and S1P. These cells may
play an important role in driving immune responses in the MLN and their
manipulation could lead to advances in controlling intestinal immune responses.
2
Abstract ...................................................................................... 2
Acknowledgments ........................................................................ 11
Authors Declaration ...................................................................... 13
List of Abbreviations ..................................................................... 14
Chapter 1: Introduction ................................................................. 17
1.1 Dendritic Cells- A Brief Introduction ......................................... 17
1.2 Dendritic Cell Development .................................................... 18
1.2.1 Cytokines Involved In Dendritic Cell Development ........................ 20
1.3 Conventional Dendritic Cell Subsets .......................................... 21
1.4 Plasmacytoid Dendritic Cells ................................................... 23
1.5 Pattern Recognition By Dendritic Cells ...................................... 24
1.6 Dendritic Cell Migration ......................................................... 25
1.7 Antigen Uptake and Presentation ............................................. 28
1.8 Dendritic Cell Interactions with the Adaptive Immune Response ...... 30
1.8.1 CD4+ T Cells ..................................................................... 30
1.8.2 CD8+ T cells ..................................................................... 33
1.8.3 B Cells ............................................................................ 34
1.9 Lymphoid Tissue Development ................................................ 35
1.9.1 Lymph Node Development .................................................... 35
1.10 Splenic Development ........................................................... 39
1.11 Mesenteric Lymph Node Development ..................................... 39
1.12 Peyer’s Patch Development .................................................. 39
1.13 Development of Mucosal Associated Lymphoid Tissues ................. 41
1.14 Formation of Tertiary Lymphoid Organs ................................... 44
1.15 The Intestinal Immune System ............................................... 45
1.15.1 Antigen Uptake in the Intestine ............................................ 45
1.15.2 M Cell Mediated Antigen Uptake ........................................... 46
1.15.3 Dendritic Cell Uptake of Luminal Antigen ................................ 47
1.15.4 Antigen Uptake Via the Neonatal Fc Receptor ........................... 51
1.16 Dendritic Cell Subsets in the Intestine ..................................... 52
1.16.1 Dendritic Cell Subsets in the Lamina Propria ............................. 52
1.16.2 Functions of Dendritic Cell Subsets in the Lamina Propria ............. 53
1.16.3 Peyer’s Patch Dendritic Cells ............................................... 54
1.16.4 Functions of Peyer’s Patch Dendritic Cell Subsets ...................... 56
3
1.16.5 Dendritic Cell Subsets in Isolated Lymphoid Follicles ................... 57
1.16.6 Functions of Dendritic Cells Within Isolated Lymphoid Follicles ...... 57
1.17 Functions of Dendritic Cells in the Mesenteric Lymph Nodes ......... 58
1.17.1 Oral Tolerance ................................................................ 58
1.17.2 Functions of Retinoic Acid in Dendritic Cells ............................. 60
1.17.3 The Generation of Foxp3+ Regulatory T Cells in the Mesenteric Lymph
Node ..................................................................................... 60
1.17.4 The Induction of Gut Homing Receptors on T Cells by Dendritic Cells 61
1.17.5 Initiation of an Adaptive Immune Response By Dendritic Cells ........ 62
1.18 Hypothesis and Aims ........................................................... 64
Chapter 2: Materials and Methods ..................................................... 66
2.1 Animals ............................................................................. 66
2.2 Surgical Procedures .............................................................. 69
2.2.1 Mesenteric Lymphadenectomy ............................................... 69
2.2.2 Thoracic Duct Cannulation .................................................... 69
2.2.3 Photoconversion of Kaede Mice .............................................. 70
2.2.4 FITC Injections .................................................................. 73
2.2.5 Subserosal Injections of Evan’s Blue ........................................ 73
2.2.6 Swiss Roll Sections of Intestine .............................................. 73
2.3 Reagents ........................................................................... 74
2.4 Cell Isolation ....................................................................... 74
2.4.1 Isolation of Thoracic Duct Leukocytes ...................................... 74
2.4.2 Isolation of Small Intestine Lamina Propria Cells ......................... 74
2.4.3 Isolation of Colonic Lamina Propria Cells ................................... 75
2.4.4 Isolation of Lymph Node and Spleen Cells .................................. 75
2.5 Flow Cytometry ................................................................... 76
2.6 Induction of DSS Colitis .......................................................... 78
2.7 Adoptive Transfers ............................................................... 78
2.7.1 Proliferation Assay ............................................................. 78
2.8 Generation of Bone Marrow Dendritic Cells ................................ 79
2.9 Preparation of Blood and Tissue Samples ................................... 79
2.10 Polymerase Chain Reaction ................................................... 80
2.11 Immunohistochemical Labelling of Slides .................................. 82
2.12 Statistical Analysis .............................................................. 82
4
Chapter 3: Comparison of Dendritic Cell Migration in the Small Intestine and
Colon ........................................................................................ 83
3.1 Results .............................................................................. 84
3.1.1 Identification of Small Intestinal and Colonic Draining Mesenteric
Lymph Nodes ........................................................................... 84
3.1.2 Evaluation of Transgenic Kaede Mice to Monitor Dendritic Cell Migration
........................................................................................... 92
3.1.3 Comparison of Levels of MHCII Expression Upon Migratory and Resident
Dendritic Cell Subsets in the Mesenteric Lymph Nodes ......................... 94
3.1.4 Comparison of Dendritic Cell Subsets in the Small Intestine, Colon,
Small Intestinal and Colonic Draining Mesenteric Lymph Nodes ............... 96
3.1.5 Comparison of Antigen Presentation by Small Intestinal and Colonic
Draining Mesenteric Lymph Node Dendritic Cells in Vitro ..................... 107
3.1.6 Dendritic Cell Subsets in the Small Intestinal and Colonic Draining
Mesenteric Lymph Nodes Following Experimental Colitis ...................... 111
3.1.7 Antigen Presentation by Small Intestinal and Colonic Draining
Mesenteric Lymph Node Dendritic Cells in Vivo ................................. 118
3.2 Discussion ........................................................................ 124
3.2.1 Identification of Draining Lymph Nodes Draining the Small Intestine and
Colon ................................................................................... 124
3.2.2 Validation of Kaede Mice as a Tool to Observe DC Migration .......... 126
3.2.3 Identification of DC subsets in the Small Intestine, Colon and Draining
Lymph Nodes .......................................................................... 128
3.2.4 Thoracic Duct Cannulation of Partially Mesenteric Lymphadenectomised
Animals ................................................................................ 133
3.2.5 Effects of Experimental Colitis Upon Dendritic Cell Subsets in the Small
Intestinal or Colonic Draining Mesenteric Lymph Nodes ....................... 135
3.2.6 Comparison of Antigen Presentation By Dendritic Cell Populations from
the Small Intestinal and Colonic Draining Mesenteric Lymph Nodes ......... 136
3.3 Conclusions ...................................................................... 138
Chapter 4: Dendritic Cell Migration in Mice Lacking Peyer’s Patches and
Isolated Lymphoid Follicles ........................................................... 140
4.1 Results ............................................................................ 141
4.1.1 Genotyping of RORγtCD3ε and CD3ε Deficient Mice ..................... 141
5
4.1.2 Lymphoid Tissue Development in the Small Intestine of RORγt Deficient
Animals ................................................................................ 145
4.1.3 Dendritic Cell Subsets in Pseudo-Afferent Lymph, Small Intestine and
Colon of RORγt Deficient Mice ..................................................... 147
4.1.4 Dendritic Cell Development in the Spleen and Bone Marrow of RORγt
Deficient Mice ........................................................................ 155
4.1.5 Generation of Lymphotoxin-β Receptor Fusion Protein ................. 159
4.1.6 Lymphoid Tissue in the Intestine of Lymphotoxin-β Receptor Fusion
Protein Treated Progeny ............................................................ 161
4.1.7 Dendritic Cell Subsets in the Mesenteric Lymph Nodes, Small Intestine,
Colon and Migrating in Pseudo -Afferent Lymph of Lymphotoxin-β Receptor
Fusion Protein Treated Progeny ................................................... 164
4.1.8 Development of Lymph Nodes, Spleen and Dendritic Cells in
Lymphotoxin-β Receptor Fusion Protein Treated Progeny ..................... 173
4.2 Discussion ........................................................................ 181
4.2.1 Abrogated Lymphoid Tissue But Normal Dendritic Cell Development in
RORγtCD3ε Deficient Mice .......................................................... 181
4.2.2 Abrogated Peyer’s Patch But Normal Lymph Node and Dendritic Cell
Development in LTβR-Ig Treated Progeny ........................................ 184
4.2.3 Dendritic Cell Subsets In The Intestine of Mice Lacking Peyer’s Patches
or Peyer’s Patches and Isolated Lymphoid Follicles ............................ 186
4.3 Conclusions ...................................................................... 190
Chapter 5: Dendritic Cell Migration From Peyer’s Patches to the Mesenteric
Lymph Nodes ............................................................................ 193
5.1 Results ............................................................................ 194
5.1.1 Dendritic Cells from the Peyer’s Patches Migrate to the Mesenteric
Lymph Nodes in Kaede Mice ........................................................ 194
5.1.2 Dendritic Cells Migrating From Peyer’s Patches to the Mesenteric
Lymph Nodes Expand In Response to Flt3 Ligand ............................... 202
5.1.3 Dendritic Cells From Peyer’s Patches Migrate in Pseudo-Afferent Lymph
.......................................................................................... 205
5.1.4 Subsets of DCs Migrating From the Peyer’s Patches to the Mesenteric
Lymph Node ........................................................................... 205
6
5.1.5 Dendritic Cells from the Peyer’s Patches Migrate to the Mesenteric
Lymph Nodes Following FITC Injection ........................................... 209
5.1.6 Dendritic Cells Migrating From Peyer’s Patches to the Mesenteric
Lymph Nodes Do Not Expand In Response to R848 or Cholera Toxin ......... 212
5.1.7 Dendritic Cells Migrating From Peyer’s Patches to the Mesenteric
Lymph Nodes Are Dependent Upon CCR7 and S1PR ............................. 214
5.1.8 Subsets of DCs Migrating From the Peyer’s Patches to the Mesenteric
Lymph Node ........................................................................... 219
5.2 Discussion ........................................................................ 221
5.2.1 Dendritic Cells from the Peyer’s Patches Migrate to the Mesenteric
Lymph Nodes .......................................................................... 222
5.2.2 Dendritic Cells Migrating From Peyer’s Patches to the Mesenteric
Lymph Nodes Expand In Response to Flt3 Ligand ............................... 224
5.2.3 Proportions of Dendritic Cells Migrating From Peyer’s Patches to the
Mesenteric Lymph Nodes Do Not Increase in Response to R848 or Cholera
Toxin ................................................................................... 225
5.2.4 Dendritic Cells Migrating From Peyer’s Patches to the Mesenteric
Lymph Nodes Are Dependent Upon CCR7 and S1PR ............................. 227
5.2.5 Subsets of DCs Migrating From the Peyer’s Patches to the Mesenteric
Lymph Node ........................................................................... 229
5.3 Conclusions ...................................................................... 231
Chapter 6: Final Discussion ........................................................... 233
6.1 Conclusions ...................................................................... 239
Chapter 7: References ................................................................. 240

7
Figure 1-1 Dendritic Cell Development. ............................................... 19

Figure 1-2 Functions of Dendritic Cell Subsets ....................................... 23

Figure 1-3 Development of T helper Subsets ......................................... 32

Figure 1-4 Timeline of the Sequential Development of Secondary Lymphoid
Organs. .................................................................................. 36

Figure 1-5 Lymph Node Development. ................................................. 38

Figure 1-6 The Development of Cryptopatches and their Expansion into Isolated
Lymphoid Follicles. ................................................................... 43

Figure 2-1 Peyer’s Patch Photoconversion ............................................ 71

Figure 2-2 Small Intestine Photoconversion ........................................... 72

Figure 3-1 Subserosal Injection of Evans Blue Distinguishes Distinct Parts of the
MLN Drain the Small Intestine and Colon. ......................................... 86

Figure 3-2 DCs Originating in the Small Intestine Drain to Specific Parts of the
MLN. ..................................................................................... 89

Figure 3-3 Dendritic Cells Originating in the Colon Drain to Specific Nodes of the
MLN. ..................................................................................... 91

Figure 3-4 Photoconversion of kaede bone marrow derived DCs does not
upregulate the expression of CD80 or CD86. ...................................... 93

Figure 3-5 Migratory Photoconverted kaede Red+ DCs in the Small intestine and
Colon Express Higher Levels of MHCII in the MLN. ............................... 95

Figure 3-6 Four Distinct Subsets of DCs are Present in the Small Intestinal
Lamina Propria. ........................................................................ 98

Figure 3-7 Three Distinct Subsets of DCs are Present in the Colonic Lamina
Propria. ................................................................................. 99

Figure 3-8 Dendritic Cell Subsets in Small Intestinal Draining MLN and Colonic
Draining MLN. ......................................................................... 103

Figure 3-9 Expression of CX3CR1 on Migratory and Resident Dendritic Cell
Subsets in siMLN and coMLN. ....................................................... 104

Figure 3-10 Small Intestine and Colon with Draining Mesenteric Lymph Nodes
Normally Removed Following Mesenteric Lymphadenectomy. ................ 105

Figure 3-11 Dendritic Cell Subsets in the Lymph of siMLNx and coMLNx Mice. 106

Figure 3-12 Migratory Dendritic Cells From the Small Intestine and Colon are
Able to Present Antigen to Naïve T Cells. ........................................ 108

Figure 3-13 Migratory Dendritic Cells From the Small Intestine and Colon are
Able to Induce CCR9 Expression in Responding T Cells. ........................ 110

Figure 3-14 Dendritic Cell Populations are Comparable in Small Intestinal and
Colonic Draining MLN During and After Experimental Colitis. ................. 113

Figure 3-15 Migratory Dendritic Cell Subsets are Comparable in Small Intestinal
and Colonic Draining MLN During and After Experimental Colitis ............ 114

Figure 3-16 Resident Dendritic Cell Subsets are Comparable in Small Intestinal
and Colonic Draining MLN During and After Experimental Colitis ............ 115

8
Figure 3-17 CD64+CD11b+CX3CR1+ Cells are Unchanged in Small Intestinal and
Colonic Draining MLN During and After Experimental Colitis. ................. 117

Figure 3-18 Orally Administered Antigen Preferentially Causes Proliferation of
CD4+ T Cells in Small Intestinal Draining Lymph Nodes. ........................ 120

Figure 3-19 Orally Administered Antigen Causes Proliferation of CD8α+ T Cells
Specifically in Small Intestinal Draining Lymph Nodes. ........................ 121

Figure 3-20 Small Intestinal Derived Ovalbumin is Only Presented to T cells in
Small intestinal Draining Lymph Nodes ........................................... 123

Figure 4-1 The Absence of CD3+ Lymphocytes in blood samples from RORγtCD3ε
and CD3ε deficient mice. ........................................................... 142

Figure 4-2 The Presence of Neomycin and RORγt Protein in RORγCD3ε and CD3ε
Deficient mice. ....................................................................... 144

Figure 4-3 Organised Lymphoid Structures Are Absent From the Small Intestine
of RORγt Deficient Mice. ............................................................ 146

Figure 4-4 CD103-CD11b- DCs Are Missing From The Lymph of RORγtCD3ε-
Deficient Mice ........................................................................ 148

Figure 4-5 Expression of CD8α on Dendritic Cell Subsets in Lymph. ............. 149

Figure 4-6 CD103-CD11b- DCs Are Missing From Small Intestinal Lamina Propria of
RORγt Deficient Mice. ............................................................... 151

Figure 4-7 Expression of CD8α on Dendritic Cell Subsets in Small Intestinal
Lamina Propria. ....................................................................... 152

Figure 4-8 CD103-CD11b- DCs Are Fewer In The Colonic Lamina Propria of WT,
CD3ε and RORγtCD3ε Deficient Mice. ............................................. 154

Figure 4-9 Characterisation of Cell Subsets in the Spleen of WT, CD3ε and
RORγtCD3ε Deficient Mice. ......................................................... 157

Figure 4-10 Characterisation of Cell Subsets Generated from Bone Marrow of
WT, CD3ε and RORγtCD3ε Deficient Mice After Culture with Flt3L. .......... 158

Figure 4-11 SDS-PAGE Gel of Purified LTβR-Ig. ...................................... 160

Figure 4-12 The Concentration of Purifed LTβR-Ig by Bradford Assay. .......... 160

Figure 4-13 Progeny of Mice Injected With LT-βR-Ig at Embryonic Day 14 and 16
Lack Peyer’s Patches But Retain Mesenteric Lymph Nodes. ................... 162

Figure 4-14 Peyer’s Patches Are Absent From the Small Intestine of LTβR-Ig
Treated Progeny. ..................................................................... 163

Figure 4-15 Four Subsets of Dendritic Cells DCs Are Present In The Lymph of
Mice Treated in Utero With LTβR-Ig. .............................................. 166

Figure 4-16 Characterisation of Cell Subsets in the MLN of WT mice and Mice
Treated with LTβR-Ig in Utero. .................................................... 167

Figure 4-17 Characterisation of Migratory and Blood Derived Dendritic Cell
Subsets in the MLN of WT and LTβR-Ig Progeny. ................................ 168

Figure 4-18 Characterisation of Dendritic Cells in Small Intestinal Draining and
Colonic Draining MLN of WT and LTβR-Ig-treated Progeny. ................... 169

9